Thromboembolic diseases remain the leading cause of death in developed countries despite the availability of anticoagulants such as warfarin (COUMADIN®), heparin, low molecular weight heparins (LMWH), synthetic pentasaccharide factor Xa inhibitors, direct thrombin inhibitors such as Bivalrudin, and antiplatelet agents such as integrin αIIbβ3 inhibitors, aspirin, clopidogrel (PLAVIX®), and Vorapaxar (Zontivity®). Additionally, current anti-platelet therapies have limitations including increased risk of bleeding as well as partial efficacy (relative cardiovascular risk reduction in the 20 to 30% range). Thus, there is an unmet medical need for safe and efficacious oral or parenteral antithrombotics for the prevention and treatment of a wide range of thromboembolic disorders.
Thrombin is a protease at the center of coagulation. Activation of platelets by thrombin, the terminal product of the coagulation cascade, is an essential component of the hemostatic response. In addition to the activation of coagulation factors and fibrinogen, thrombin regulates cellular activities through stimulation of the G-protein coupled protease activated receptors (PARs). These receptors are activated by cleavage by thrombin, and in a unique mechanism, the new amino terminus is the activating “tethered ligand”. This causes irreversible activation of the receptors. In humans, platelets express two PARs, PAR1 and PAR4. PAR1 is ubiquitously expressed, and PAR1 signaling underlies not only coagulation, but also inflammation, pain, healing and cancer metastasis, while the expression of PAR4 is much more restricted, mainly to platelets and expression in certain brain areas and vascular beds after stress.
PAR1 is the “high affinity” thrombin receptor, while PAR4 requires much higher thrombin for activation, levels probably only seen in a platelet clot. Due to this difference in affinity, PAR1 and PAR4 are engaged in a progressive manner, with PAR1 activated at low thrombin concentrations and PAR4 recruited at higher thrombin concentrations. Because of the delay in activation we hypothesize that PAR4 antagonism might not affect hemostasis as potently and thus may be a better therapeutic target than PAR1.
Inhibitors of PAR1 have been investigated extensively, and several compounds, including Vorapaxar and atopaxar have advanced into late stage clinical trials. Recently, in the TRACER phase III trial among non-ST-segment elevation acute coronary syndromes (ACS) patients, Vorapaxar did not significantly reduce the primary composite endpoint, and in fact was halted early due to a significant increase in the risk of major bleeding, including intracranial hemorrhage (Tricoci, P. et al, N. Eng. J. Med., 366(1):20-33 (2012). However, among non-ST-segment ACS patients undergoing CABG specifically, Vorapaxar was associated with a significant reduction in ischemic events and no significant increase in major CABG-related bleeding (Whellan D J et al, J Am Coll Cardiol., 63(11): 1048-57(2014). The TRA 2P-TIMI 50 trial demonstrated that in patients with myocardial infarction, Vorapaxar reduced the risk of cardiovascular death or ischaemic events with a significant increase in moderate to severe bleeding when added to the standard anti-platelet therapy (Scirica B M et al, Lancet., 380(9850): 1317-24 (2012). Similar results were collected among patients with peripheral artery disease demonstrating significant beneficial effects on limb ischemia and peripheral revascularization with increased risk of bleeding (Bonaca M P et al, Circulation, 127(14): 1522-9 (2013). However, among patients with prior ischemic stroke adding Vorapaxar to the standard of care increased the risk of intracranial hemorrhage without improvement in major vascular events (Morrow D A et al, Stroke 44(3):691-8 (2013). Therefore, even though the PAR-1 antagonist Vorapaxar (Zontivity™) was approved by the FDA as the first in class protease activated receptor antagonist, its potential application is severely limited by the bleeding side effects and increased risk of hemorrhagic stroke. The inability of PAR1-inhibited platelets to sense and respond to low levels of thrombin is likely a contributing factor to the bleeding events observed in patients using Vorapaxar (Zontivity™).
Mice express PAR3 and PAR4 on their platelet surface. PAR3 itself does not signal but serves as a cofactor, enhancing PAR4 activation by thrombin (Kahn, M. L., et al. (1998) A dual thrombin receptor system for platelet activation, Nature 394, 690-694; Nakanishi-Matsui, M., et al. (2000) PAR3 is a cofactor for PAR4 activation by thrombin, Nature 404, 609-613.) Intriguingly, PAR4−/− mice are protected from thrombosis and cerebral ischemia/reperfusion injury, have prolonged tail bleeding times, but no bleeding disorder. These phenotypes in mice are consistent with the fact PAR4 knockout results in a platelet that is incapable of responding to thrombin (Sambrano, G. R., et al. (2001) Role of thrombin signalling in platelets in haemostasis and thrombosis, Nature 413, 74-78.). These data combined with the fact that PAR4 is the lower affinity receptor and thus not engaged until later stages of hemostasis and possibly thrombosis, suggest that PAR4 is an attractive target for a safer anti-platelet therapy in thrombosis and cerebrovascular injury. Fortunately in humans both PAR1 and PAR4 are expressed on platelets, allowing for a dual thrombin receptor system. This presents a unique pharmacological opportunity, allowing inhibition of one PAR to preserve the platelet response to thrombin through the other, thereby perturbing but not eliminating thrombin-mediated platelet signaling. The physiologic role for PAR4 in hemostasis and thrombosis has not been fully established in humans; however, the pharmacodynamics of this receptor suggests that it may contribute to the pathophysiology of thrombosis. The signaling effectors engaged by PAR1 and PAR4 are essentially redundant; however responses display differences in both magnitude and kinetics. PAR1 signaling quickly desensitizes after activation while PAR4 signaling persists (Falker, K., et al. (2011) Protease-activated receptor 1 (PAR1) signalling desensitization is counteracted via PAR4 signalling in human platelets, The Biochemical Journal 436, 469-480.). This is reflected in the Ca2+ response which is rapid and transient downstream of PAR1, but slow and sustained downstream of PAR4 (Covic, L., et al. (2000) Biphasic kinetics of activation and signaling for PAR1 and PAR4 thrombin receptors in platelets, Biochemistry 39, 5458-5467.). Similar profiles were obtained for Protein Kinase C (PKC) and Rhokinase mediated phosphorylation with stronger and more sustained activity downstream of PAR4 activation. Importantly, this leads to greater integrin activation and more secretion through PAR4, which is capable of overcoming the PAR1 response when fully engaged (Duvernay, M., et al. (2013). Protease-activated receptor (PAR) 1 and PAR4 differentially regulate factor V expression from human platelets, Molecular Pharmacology 83, 781-792; Vretenbrant, K., et al. (2007). Platelet activation via PAR4 is involved in the initiation of thrombin generation and in clot elasticity development, Thrombosis and haemostasis 97, 417-424.). Due to the higher affinity of PAR1 for thrombin there is a sequential nature to PAR engagement by thrombin on the human platelet, with PAR1 activation prior to PAR4 as thrombin concentrations rise in response to vascular injury. With the redundancy in signaling, full inhibition of PAR4 activity would at best result in a partial reduction in the magnitude and duration of the platelet response to thrombin due to intact PAR1 signaling. In addition, inhibitors with partial antagonism of the PAR4 receptor would be expected to partly preserve PAR-4 mediated platelet signaling activation, therefore providing a means to further modulate efficacy and potency via PAR4 inhibition. In summary, based on the latent engagement and activation of PAR4 at higher thrombin concentrations and the observations from the animal studies described, we hypothesize that PAR4 antagonism may be a safer and better therapeutic approach than PAR1 to treat thrombotic disorders and cerebrovascular injury and potentially primary and secondary prevention.
There are several early reports of preclinical studies of PAR4 inhibitors. Lee, F-Y. et al., “Synthesis of 1-Benzyl-3-(5′-hydroxymethyl-2′-furyl)indazole Analogues as Novel Antiplatelet Agents”, J. Med. Chem., 44(22):3746-3749 (2001) discloses in the abstract that the compound:
“was found to be a selective and potent inhibitor or protease-activated receptor type 4 (PAR4)-dependent platelet activation.”
YD-3 was also referenced in Wu, C-C. et al, “Selective Inhibition of Protease-activated Receptor 4-dependent Platelet Activation by YD-3”, Thromb. Haemost., 87: 1026-1033 (2002). Also, see Chen, H. S. et al, “Synthesis and platelet activity”, J. Bioorg. Med. Chem., 16: 1262-1278 (2008).
EP1166785 A1, EP0667345, WO 2013/163248 and WO 2013/163279, all incorporated herein by reference, disclose various compounds which are useful as inhibitors of platelet aggregation.
As indicated in WO 2014/173859 and WO 2015/124570, both of which are incorporated herein by reference, compounds of the present invention are useful for treating or preventing influenza virus type A infections. As indicated in WO 2015/124570, influenza is one of the most common infectious diseases in humans, occurring as seasonal epidemic and sporadic pandemic outbreaks. Annually, influenza A viruses (IAV) cause 3-5 million clinical infections and 200,000-500,000 fatal cases.
The hallmark of severe influenza virus infections is excessive inflammation of the lungs. Platelets are activated during influenza, but their role in influenza virus pathogenesis and inflammatory responses is unknown. Targeted gene deletion approaches and pharmacological interventions have been used to investigate the role of platelets during influenza virus infection in mice. Lungs of infected mice were massively infiltrated by aggregates of activated platelets. Platelet activation promoted IAV pathogenesis. Activating protease-activated receptor 4 (PAR-4), a platelet receptor for thrombin that is crucial for platelet activation, exacerbated influenza-induced acute lung injury and death. In contrast, deficiency in the major platelet receptor glycoprotein Ilia (GPIIIa) protected mice from death caused by influenza viruses, and treating the mice with a specific GPIIbllla antagonist, eptifibatide, had the same effect. Interestingly, mice treated with other anti-platelet compounds (such as antagonists of PAR-4, for example) were also protected from severe lung injury and lethal infections induced by several influenza strains. The intricate relationship between hemostasis and inflammation has major consequences in influenza virus pathogenesis, and anti-platelet drugs have been explored to develop new anti-inflammatory treatment against influenza virus infections.
Accordingly with the compounds of the present invention being antagonists of PAR-4, an object of the present invention relates to a method for the treatment of influenza A virus (IAV) infection in a subject in need thereof comprising administering the subject with a therapeutically effective amount of at least one anti-platelet agent of the present invention. As used herein, the term “influenza A virus infection” or “IAV infection” has its general meaning in the art and refers to the disease caused by an infection with an influenza A virus. In some embodiments of the invention, IAV infection is caused by influenza virus A that is HIM, H2N2, H3N2 or H5N1. As used herein, an “anti-platelet agent” refers to members of a class of pharmaceuticals that inhibit platelet function, for example, by inhibiting the activation, aggregation, adhesion or granular secretion of platelets.
As indicated in WO 2013/163279, the PAR-4 antagonists of the present invention are useful as selective inhibitors of platelet aggregation, including stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, or prodrug esters thereof.
Accordingly, in another embodiment, the present invention provides a method for the treatment of a thromboembolic disorder, wherein the thromboembolic disorder is selected from unstable angina, an acute coronary syndrome, atrial fibrillation, myocardial infarction, transient ischemic attack, stroke, atherosclerosis, peripheral occlusive arterial disease, venous thrombosis, deep vein thrombosis, thrombophlebitis, arterial embolism, coronary arterial thrombosis, cerebral arterial thrombosis, cerebral embolism, kidney embolism, pulmonary embolism, and thrombosis resulting from medical implants, devices, or procedures in which blood is exposed to an artificial surface that promotes thrombosis. In another embodiment, the present invention provides a method for the treatment of a thromboembolic disorder, wherein the thromboembolic disorder is selected from acute coronary syndrome, stroke, venous thrombosis, atrial fibrillation, and thrombosis resulting from medical implants and devices.
In another embodiment, the present invention provides a method for the primary prophylaxis of a thromboembolic disorder, wherein the thromboembolic disorder is selected from unstable angina, an acute coronary syndrome, atrial fibrillation, myocardial infarction, ischemic sudden death, transient ischemic attack, stroke, atherosclerosis, peripheral occlusive arterial disease, venous thrombosis, deep vein thrombosis, thrombophlebitis, arterial embolism, coronary arterial thrombosis, cerebral arterial thrombosis, cerebral embolism, kidney embolism, pulmonary embolism, and thrombosis resulting from medical implants, devices, or procedures in which blood is exposed to an artificial surface that promotes thrombosis. In another embodiment, the present invention provides a method for the primary prophylaxis of a thromboembolic disorder, wherein the thromboembolic disorder is selected from acute coronary syndrome, stroke, venous thrombosis, and thrombosis resulting from medical implants and devices.
In another embodiment, the present invention provides a method for the secondary prophylaxis of a thromboembolic disorder, wherein the thromboembolic disorder is selected from unstable angina, an acute coronary syndrome, atrial fibrillation, recurrent myocardial infarction, transient ischemic attack, stroke, atherosclerosis, peripheral occlusive arterial disease, venous thrombosis, deep vein thrombosis, thrombophlebitis, arterial embolism, coronary arterial thrombosis, cerebral arterial thrombosis, cerebral embolism, kidney embolism, pulmonary embolism, and thrombosis resulting from medical implants, devices, or procedures in which blood is exposed to an artificial surface that promotes thrombosis. In another embodiment, the present invention provides a method for the secondary prophylaxis of a thromboembolic disorder, wherein the thromboembolic disorder is selected from acute coronary syndrome, stroke, atrial fibrillation and venous thrombosis.
It is noted that thrombosis includes vessel occlusion (e.g., after a bypass) and reocclusion (e.g., during or after percutaneous transluminal coronary angioplasty). The thromboembolic disorders may result from conditions including but not limited to atherosclerosis, surgery or surgical complications, prolonged immobilization, arterial fibrillation, congenital thrombophilia, cancer, diabetes, effects of medications or hormones, and complications of pregnancy.
Thromboembolic disorders are frequently associated with patients with atherosclerosis. Risk factors for atherosclerosis include but are not limited to male gender, age, hypertension, lipid disorders, and diabetes mellitus. Risk factors for atherosclerosis are at the same time risk factors for complications of atherosclerosis, i.e., thromboembolic disorders.
Similarly, arterial fibrillation is frequently associated with thromboembolic disorders. Risk factors for arterial fibrillation and subsequent thromboembolic disorders include cardiovascular disease, rheumatic heart disease, nonrheumatic mitral valve disease, hypertensive cardiovascular disease, chronic lung disease, and a variety of miscellaneous cardiac abnormalities as well as thyrotoxicosis.
Diabetes mellitus is frequently associated with atherosclerosis and thromboembolic disorders. Risk factors for the more common type 2 include but are not limited to family history, obesity, physical inactivity, race/ethnicity, previously impaired fasting glucose or glucose tolerance test, history of gestational diabetes mellitus or delivery of a “big baby”, hypertension, low HDL cholesterol, and polycystic ovary syndrome.
Thrombosis has been associated with a variety of tumor types, e.g., pancreatic cancer, breast cancer, brain tumors, lung cancer, ovarian cancer, prostate cancer, gastrointestinal malignancies, and Hodgkins or non-Hodgkins lymphoma. Recent studies suggest that the frequency of cancer in patients with thrombosis reflects the frequency of a particular cancer type in the general population. (Levitan, N. et al., Medicine (Baltimore), 78(5):285-291 (1999); Levine M. et al, N. Engl. J. Med., 334(11):677-681 (1996); Blom, J. W. et al, JAMA, 293(6):715-722 (2005).) Hence, the most common cancers associated with thrombosis in men are prostate, colorectal, brain, and lung cancer, and in women are breast, ovary, and lung cancer. The observed rate of venous thromboembolism (VTE) in cancer patients is significant. The varying rates of VTE between different tumor types are most likely related to the selection of the patient population. Cancer patients at risk for thrombosis may possess any or all of the following risk factors: (i) the stage of the cancer {i.e., presence of metastases), (ii) the presence of central vein catheters, (iii) surgery and anticancer therapies including chemotherapy, and (iv) hormones and antiangiogenic drugs. Thus, it is common clinical practice to dose patients having advanced tumors with heparin or low molecular heparin to prevent thromboembolic disorders. A number of low molecular weight heparin preparations have been approved by the FDA for these indications.
Gamma-thrombin is a proteolytic product of alpha-thrombin cleavage by a serine protease. Cleavage results in the disruption of exosite I which interacts directly with the hirudin-like domain of its primary substrates fibrinogen and protease-activated receptor 1 (PAR1). As a result, gamma-thrombin selectively cleaves PAR4 in the nanomolar range, leaving PAR1 intact. See Soslau, G., Class, R., Morgan, D. A., Foster, C., Lord, S. T., Marchese, P., and Ruggeri, Z. M. (2001) Unique pathway of thrombin-induced platelet aggregation mediated by glycoprotein Ib, The Journal of biological chemistry 276, 21173-21183. Gamma-thrombin (Enzyme Research Laboratories, South Bend, Ind.) is produced by trypsin-sepharose cleavage of alpha-thrombin. To neutralize alpha-thrombin contamination, stock solutions of gamma-thrombin are preincubated with 1 unit/mL hirudin prior 100 fold dilution into the sample for platelet stimulation. This concentration of hirudin is capable of abolishing alpha-thrombin activity with no effect on the activity of gamma-thrombin. See Soslau, G., Goldenberg, S. J., Class, R., and Jameson, B. (2004) Differential activation and inhibition of human platelet thrombin receptors by structurally distinct alpha-, beta- and gamma-thrombin, Platelets 15, 155-166. Single point screens and concentration response curves are conducted with 316 nanomolar or 100 nanomolar gamma-thrombin using PAC1 and CD61p binding.
Embodiments of the present invention are also useful in reducing injury from myocardial ischemia/reperfusion. Decreased PAR1 mRNA and increased PAR4 mRNA detection in the rat brain after endothelin injection into the middle cerebral artery. SeeRohatgi, T., Sedehizade, F., Sabel, B. A., and Reiser, G. (2003) Protease-activated receptor subtype expression in developing eye and adult retina of the rat after optic nerve crush, Journal of neuroscience research 73, 246-254. Enhanced immunohistochemical labeling of PAR4 after endothelin injection into the middle cerebral artery in the border zone and the infarct zone. See Henrich-Noack, P., Riek-Burchardt, M., Baldauf, K., Reiser, G., and Reymann, K. G. (2006) Focal ischemia induces expression of protease-activated receptor1 (PAR1) and PAR3 on microglia and enhances PAR4 labeling in the penumbra, Brain research 1070, 232-241. Inhibition of PAR4 (P4pal 10 and trans-cinnamoyl-YPGKF) reduced infarct size in a rat model of myocardial ischemia/repurfusion injury. See Strande, J. L., Hsu, A., Su, J., Fu, X., Gross, G. J., and Baker, J. E. (2008) Inhibiting protease-activated receptor 4 limits myocardial ischemia/reperfusion injury in rat hearts by unmasking adenosine signaling, The Journal of pharmacology and experimental therapeutics 324, 1045-1054 PAR4 deficiency resulted in an 80% reduction in infarct volume following transient middle cerebral artery occlusion (MCAO). Mao, Y., Zhang, M., Tuma, R. F., and Kunapuli, S. P. (2010) Deficiency of PAR4 attenuates cerebral ischemia/reperfusion injury in mice, Journal of cerebral blood flow and metabolism: official journal of the International Society of Cerebral Blood Flow and Metabolism 30, 1044-1052. PAR4 deficiency resulted in reduced infarct size and more robust functional recovery in in vivo and ex vivo models of myocardial ischemia reperfusion injury. See Kolpakov, M. A., Rafiq, K., Guo, X., Hooshdaran, B., Wang, T., Vlasenko, L., Bashkirova, Y. V., Zhang, X., Chen, X., Iftikhar, S., Libonati, J. R., Kunapuli, S. P., and Sabri, A. (2016) Protease-activated receptor 4 deficiency offers cardioprotection after acute ischemia reperfusion injury, Journal of molecular and cellular cardiology 90, 21-29. PAR4 mediates proapoptotic signaling on isolated cardiomyocytes.
Embodiments of the present invention are also useful in the treatment of inflammation. For example, activation of PAR4 on epithelial cells leads to shape change, increased permeability, endothelial-dependent vasodilation and edema. PAR4 activation also induces Von Willebrand Factor and P-selectin expression in addition to cytokine production which is known to recruit platelets and leukocytes to sites of inflammation. See Subramaniam, M., Frenette, P. S., Saffaripour, S., Johnson, R. C., Hynes, R. O., and Wagner, D. D. (1996) Defects in hemostasis in P-selectin-deficient mice, Blood 87, 1238-1242; Frenette, P. S., Mayadas, T. N., Rayburn, H., Hynes, R. O., and Wagner, D. D. (1996) Susceptibility to infection and altered hematopoiesis in mice deficient in both P- and E-selectins, Cell 84, 563-574; and Hattori, R., Hamilton, K. K., Fugate, R. D., McEver, R. P., and Sims, P. J. (1989) Stimulated secretion of endothelial von Willebrand factor is accompanied by rapid redistribution to the cell surface of the intracellular granule membrane protein GMP-140, The Journal of biological chemistry 264, 7768-7771.
Leukocyte recruitment to endothelial cells at the site of inflammation is a hallmark of the inflammatory response. The first indicator for PAR4 role in inflammation comes from the imaging of leukocyte recruitment in an in vivo intravital microscopy system. Topical administration of thrombin to the mesenteric venule results in increased leukocyte rolling and adhesion. These results were recapitulated with PAR4-AP but not PAR1-AP. Moreover, intraperitoneal injection of PAR4-AP caused significant increase in extravascular leukocyte migration into the peritoneal cavity. See Vergnolle, N., Derian, C. K., D'Andrea, M. R., Steinhoff, M., and Andrade-Gordon, P. (2002) Characterization of thrombin-induced leukocyte rolling and adherence: a potential proinflammatory role for proteinase-activated receptor-4, Journal of immunology 169, 1467-1473. More recently, the PAR4 antagonist YPGKF-NH 2 (tcY-NH2) was shown to inhibit neutrophil migration into CXCL8, carrageenan (Cg), PAR4-AP, or Trypsin injected pleural cavities of mice. See Gomides, L. F., Lima, O. C., Matos, N. A., Freitas, K. M., Francischi, J. N., Tavares, J. C., and Klein, A. (2014) Blockade of proteinase-activated receptor 4 inhibits neutrophil recruitment in experimental inflammation in mice, Inflammation research: official journal of the European Histamine Research Society . . . [et al.] 63, 935-941; Gomides, L. F., Duarte, I. D., Ferreira, R. G., Perez, A. C., Francischi, J. N., and Klein, A. (2012) Proteinase-activated receptor-4 plays a major role in the recruitment of neutrophils induced by trypsin or carrageenan during pleurisy in mice, Pharmacology 89, 275-282. tcY-NH2 also blocks eosinophil recruitment after intrapleural injection of the chemokine Eotaxin-1. See Braga, A. D., Miranda, J. P., Ferreira, G. M., Bilheiro, R. P., Duarte, I. D., Francischi, J. N., and Klein, A. (2010) Blockade of proteinase-activated receptor-4 inhibits the eosinophil recruitment induced by eotaxin-1 in the pleural cavity of mice, Pharmacology 86, 224-230.
Additionally, inflammatory arthritis is a localized pathology characterized by increased thrombin generation and coagulation factor activation, even compared to osteoarthritis (mechanical as opposed to immune induced inflammation). See So, A. K., Varisco, P. A., Kemkes-Matthes, B., Herkenne-Morard, C., Chobaz-Peclat, V., Gerster, J. C., and Busso, N. (2003) Arthritis is linked to local and systemic activation of coagulation and fibrinolysis pathways, J Thromb Haemost 1, 2510-2515. Tissue factor levels and activity are increase in the synovial fluid of patients with rheumatoid arthritis. See Busso, N., Morard, C., Salvi, R., Peclat, V., and So, A. (2003) Role of the tissue factor pathway in synovial inflammation, Arthritis and rheumatism 48, 651-659. Recent work seeking to describe how excess thrombin contributes to disease progression has indicated PAR4 as a possible modulator. PAR4 knock out drastically reduces the inflammatory response in a mouse model of tissue factor induced inflammatory arthritis. See Busso, N., Chobaz-Peclat, V., Hamilton, J., Spee, P., Wagtmann, N., and So, A. (2008) Essential role of platelet activation via protease activated receptor 4 in tissue factor-initiated inflammation, Arthritis research & therapy 10, R42. Similar results are obtained in a kaolin/carrageenan induced model of inflammatory arthritis where PAR4 inhibition with a pepducin antagonist reduces indices of inflammatory arthritis. In the same study, direct injection of PAR4-AP induced joint swelling and hyperalgesia. See McDougall, J. J., Zhang, C., Cellars, L., Joubert, E., Dixon, C. M., and Vergnolle, N. (2009) Triggering of proteinase-activated receptor 4 leads to joint pain and inflammation in mice, Arthritis and rheumatism 60, 728-737. Similar results were obtained in general rat hindpaw carrageenan injected model of inflammation. Pepducin P4pal-10 and palmitoly-SGRRYGHALR both reduced edema and granulocyte recruitment and direct injection of PAR4-AP caused edema and granulocyte recruitment. The indicated physiologic effect of PAR4 activation can be mediated through either platelets or neutrophils, both of which express PAR4. Yet, depletion of neutrophils significantly reduced PAR4-AP induced edema. See Houle, S., Papez, M. D., Ferazzini, M., Hollenberg, M. D., and Vergnolle, N. (2005) Neutrophils and the kallikrein-kinin system in proteinase-activated receptor 4-mediated inflammation in rodents, British journal of pharmacology 146, 670-678. These results indicate PAR4 activation on neutrophils as a pivotal event in inflammatory responses in the joint.
The compounds of the present invention are also useful in the treatment of sepsis. Sepsis is characterized by systemic inflammation and disseminated intravascular coagulation (DIC), the so-called Schwartzman reaction. In an endotoxin induced model of murine sepsis, P4pal-10 (PAR4 inhibitor) but not Plpal-12 (PAR1 inhibitor) reduced several indicators of organ failure and neutrophil influx, which are characteristic of the pathology. See Slofstra, S. H., Bijlsma, M. F., Groot, A. P., Reitsma, P. H., Lindhout, T., ten Cate, H., and Spek, C. A. (2007) Protease-activated receptor-4 inhibition protects from multiorgan failure in a murine model of systemic inflammation, Blood 110, 3176-3182. The Schwartzman reaction involves the interplay between immune and coagulation systems. PAR4 is expressed on neutrophils and platelets, which represent sentinels of each system respectively. In order to determine where PAR4 inhibition was exerting its effect on the system, mice were depleted of either neutrophils or platelets. In neutrophil depleted mice the protective effects of P4pal-10 were abolished indicating that PAR4 activation on neutrophils is pivotal to organ damage associated with DIC.
The compounds of the present invention are also useful in the treatment of inflammatory bowel disease. Inflammatory bowel diseases such as Chrone's and ulcerative colitis are characterized by increased permeability of the intestinal epithelial barrier, penetration of luminal products, and an immune response characterized by neutrophil invasion and cytokine driven inflammation. See Weber, C. R., and Turner, J. R. (2007) Inflammatory bowel disease: is it really just another break in the wall?, Gut 56, 6-8; Podolsky, D. K. (2002) Inflammatory bowel disease, The New England journal of medicine 347, 417-429. Fecal supernatants from patients with ulcerative colitis were shown to have abnormally high serine protease activity. See Gecse, K., Roka, R., Ferrier, L., Leveque, M., Eutamene, H., Cartier, C., Ait-Belgnaoui, A., Rosztoczy, A., Izbeki, F., Fioramonti, J., Wittmann, T., and Bueno, L. (2008) Increased faecal serine protease activity in diarrhoeic IBS patients: a colonic lumenal factor impairing colonic permeability and sensitivity, Gut 57, 591-599. Both PAR4 and its neutrophil derived specific agonist Cathepsin G are over-expressed in human colonic biopsies. Intracolonic infusion of fecal supernatants from ulcerative colitis patients induced para cellular permeability that was blocked with a PAR4 antagonist or a Cathepsin G inhibitor. See Dabek, M., Ferrier, L., Roka, R., Gecse, K., Annahazi, A., Moreau, J., Escourrou, J., Cartier, C., Chaumaz, G., Leveque, M., Ait-Belgnaoui, A., Wittmann, T., Theodorou, V., and Bueno, L. (2009) Luminal cathepsin G and protease-activated receptor 4: a duet involved in alterations of the colonic epithelial barrier in ulcerative colitis, The American journal of pathology 175, 207-214. In the same study the PAR4-AP was capable of inducing similar para cellular permeability.